BIOCHEMISTRY & BIOMEDICAL SCIENCES

Multiple Choice Question with answers on Chlamydia trachomatis & other Chlamydiaceae

1) Which of the following Chlamydia spp are human pathogens? Select from all the options given below
a) Chlamydia pecorum
b) Chlamydia psittaci
c) Chlamydia trachomatis
d) Chlamydia pnuemoniae

2) What serotypes of Chlamydia trachomatis is associated with inclusion conjunctivitis seen in adults and neonates?
a) Serotypes D to K
b) Serotypes L1,2 and 3
c) Serotypes A, B, and C
d) Serotypes A, B, B and C

3) What type of laboratory methods are used for the diagnosis of genital Chlamydia trachomatis infections? Select all that applies
a) Specimen culture in McCoy cell tissue culture
b) Direct fluorescent antibody and Enzyme-linked immunoassay
c) Serological tests
d) Nucleic acid amplification test


4) Chlamydia trachomatis is transmitted from infected mother to baby through the birth canal and can cause neonatal inclusion conjunctivitis, how many days after the baby is born symptoms in eyes start to develop?
a) After 24 hours
b) Within 48 hours
c) After 6 hours
d) Within 7 to 15 days

5) All of the following statements are true about the antigens of Chlamydia spp, Except?
a) Genus-specific antigens are common to all Chlamydia
b) Only Chlamydia trachomatis have genus-specific antigens
c) Species-specific antigens are present in the outer membrane of Chlamydia spp
d) None of the above

6) Which of the following statements is NOT the distinguishing features of the family Chlamydiaceae?
a) They are seen on Gram stain
b) They are obligate intracellular bacteria
c) They cannot make ATP
d) They consist of two forms elementary and reticulate body

7) All of the statements given below are correct about Chlamydia psittaci, EXCEPT?
a) It is a primary pathogen of birds
b) It can cause respiratory infections in human
c) It is usually resistant to sulfonamides
d) It only infects birds like parrots and cockatoos

c) Validation of c90rf72 gene silencing and AAV5 mediated gene delivery in HEK cells and iPSC neurons show successful transduction and expression of microRNA and decrease in c9orf72 RNA and RNA foci

 d) Proof-of-concept of AVV5 mediated gene delivery shows transduction of AAV5 vector on various brain tissue, expression of microRNA transgene and decreased nuclear and cytoplasmic RNA foci in c9orf72_3 transgenic mice.
d) The durability of the transgene expression in non-human primates and clinical subjects

Multiple Choice Question on T cell Development, Differentiation and Activation

1) T cells (T lymphocytes) are crucial in recognition of antigens presented by self-MHC. The T cell progenitors undergo proliferation and differentiation in thymus and form a mature T cell. Which of the following is the origin for T cell progenitors?
a) Thymus
b) Hepatocytes
c) Bone marrow
d) None of the above

2) What is the characteristic feature of progenitor T cells that have migrated to the thymus?
a) They express T cell receptor/CD3 complex
b) They express CD28 cell adhesion molecule
c) They express CD4 or CD8 co-receptor
d) None of the above

3) Which of the following cell adhesion molecule is present in the T cell progenitors is required for homing of these cells into thymus?
a)CD25
b) CD44
c) IL-2
d) CTLA-4

4) Pre- T cell receptor complex consists of the β chain of TCR & CD3 molecules that are formed during the proliferation of T cells. The function of the pre-TCR complex include except
a) signal productive rearrangement of TCR β for further proliferation
b) suppress further rearrangement of β chain (allelic exclusion)
c) prepare cells for rearrangement of α chain
d) commit T cells for CD4 or CD8 positive T cells

5) Which of the following is the process for T cell development and maturation?
a) Rearrangement of T cell receptor and expression of coreceptors
b) Positive selection of thymocytes bearing receptors that are capable of binding to self-MHC molecules (MHC restriction)
c) Negative selection ensure the affinity receptor self MHC or MHC antigen complex is eliminated (self-tolerance)
d) All of the above

6) During the differentiation of T lymphocytes, the double positive cells are directed to become CD4 + T cell & CD8+ T cell which are MHC II & MHC I restricted respectively. Which of the following process may be correct for T cell differentiation?
a) Intrinsic model- multiple interactions of MHC with double positive (CD4+ & CD8+) instruct the cell to differentiate
b) Stochastic model- CD4 or CD8 expression in switched of randomly
c) Both A and B
d) None of the above

7) The activation of T cell requires the interaction of MHC/peptide of TCR/CD3 complex activation require expression of
a) Transcription factors such as c-Fos, c-Myc, c-Jun etc.
b) Interleukins such as IL-2, IL-3, and IL-6
c) Adhesion molecules such as CD28, CTLA-4
d) All of the above

8) Which of the following cytoplasmic tail of CD4 or CD8 coreceptors are required for phosphorylation of ITAM present of CD3 molecules?
a) Lck
b) ZAP70
c) LAD
d) None of the above

9) The cell surface proteins on T cell bind to B7 on the antigen presenting cells and serve as a secondary signal. Which of the following is true regarding the secondary signal?
a) CD28 is a protein that binds to B7 on APC that acts as costimulatory signals for T cell activation
b) CTLA-4 is a protein that binds to B7 on APC that acts as a suppressor signal for T cell activation
c) Both
d) None

10) Which of the cytokines function in an autocrine manner and induces T cell proliferation after engagement of TCR with antigens interaction and presence of secondary signal?
a) IL-1
b) IL-2
c) IL-3
d) IL-4

11) Cytokines such as IL-2, IFN-γ, TNF-β, play an important role in cell-mediated cytotoxicity and delay hypersensitivity. Which of the following subset of T helper cells are involved in the process
a) T helper 1 CD4+
b) T helper 2 CD4+
c) T helper 1 CD8+
d) T helper 2 CD8+

12) Cytokines such as IL-4, IL-5, IL-6 & IL-10 play an important role in B cell activation and humoral immune response
a) T helper 1 CD4+
b) T helper 2 CD4+
c) T helper 1 CD8+
d) T helper 2 CD8+

13) The regulatory or suppressor T cells express the specific cell surface marker for its function. It is
a) CD4+ CD25+
b) CD4+ CD25-
c) CD4 - CD8 - CD25 +
d) CD4 + CD8 + CD25 +

Multiple Choice Question on Treponema Pallidum & Other Spirochetes

1) Which of the following is not the general characteristics of spirochetes?
a) They are gram-negative helical bacteria
b) They are motile and have periplasmic flagella (endoflagella)
c) They reproduce by transverse binary fusion
d) They are obligate aerobes

2) Which of the following subspecies of Treponema pallidum causes endemic syphilis?
a) Treponema carateum
b) Treponema endemicum
c) Treponema pertenue
d) None of the above

3) The RPR (Rapid Plasma Reagin) is a nontreponemal test method used as a screening test for syphilis, which of the following statements is correct about the test?
a) It is an expensive method
b) It often shows a positive result with primary syphilis
c) It often shows a false positive result for syphilis
d) It is a specific test for Treponema pallidum


4) A 30 years old man infected with syphilis develops a skin lesion on his prepuce, the lesion is oval in shape and is painless. What is the specific name for this type of lesions?
a) Nodules
b) Papule
c) Eczema
d) Hard chancre

5) Epidemic Relapsing fever in humans is caused by the infected body louse which acts as a vector and is transmitted to humans, which of the following is the most likely spirochetes?
a) Borrelia recurrentis
b) Leptospira interrogans
c) Borrelia burgdorferi
d) Borrelia hermsii

6) These bacteria are motile, found worldwide and infect kidneys and lever which can lead to infectious jaundice, the bacterial serovars are transmitted to humans through contact with animals and its excreta. What is the likely pathogen?
a) Borrelia burgdorferi
b) Bordetella pertusis
c) Leptospira interrogans
d) Enterococcus faecalis

7) Which of the following statements is most correct about the dark field microscopy for the diagnosis of spirochetes?
a) It is used to observe and detect thin spirochetes suspended in a liquid
b) The specimen is stained and detected under the compound microscope
c) The color of the spirochetes appears grey when observed under dark background
d) It is used to detect the spirochetes from the oral cavity, cerebrospinal fluid

8) Which of the following organisms is NOT the normal flora of the oral cavity?
a) Streptococcus mutans
b) Borrelia buccalis
c) Spirillum minor
d) Candida albicans

9) What stain is mostly used for the detection of Borrelia spp which causes relapsing fever?
a) Gram stain
b) Ziehl-Neelsen stain
c) Giemsa stain
d) Methylene blue

10) The bacteria causing Leptospirosis are difficult to isolate from urine, blood or CSF specimens by culture method, therefore a confirmation test should be done. Which of the following statement is most correct in the diagnosis of the disease?
a) RPR tests for the detection of serogroup antigens from the acute or convalescent phase
b) Antibodies are tested against the serogroup antigens from the acute or convalescent phase
c) Darkfield microscopy method is done to detect the pathogen from serum samples
d) All of the above

Anti-drug antibody (ADA) assay is a semi-quantitative bioanalytical method that detects the antibodies specific against the drug. The ADA assay utilizes the assay cut point above which the samples are considered positive. The assay cut points are established by analyzing a set of approximately 50 drug-naive individuals with an assumption that these selected samples do not contain any pre-existing antibodies.

While this is true for most ADA assays, in some instances, there is a high probability of prevalent pre-existing antibodies when a closely related antigen is exposed prior to drug administration. The pre-exposure of antigens such as adeno associated viruses (AAVs), Cas9 protein, PEG molecule account for preexisting antibodies in clinical subjects. When the preexisting antibody is prevalent in the study population, the cut point should be established carefully to enable the detection of the clinically significant ADA. Applying the recommended statistical cut point calculation method (Shankar et al, 2008) in these preexisting ADA populations may result in elevated cut points and a higher false-negative rate. To overcome this challenge, the immune-inhibition method is reported and acceptable to the FDA regulatory agencies.

Immunoinhibition method for preexisting antibodies
In the immune-inhibition method, the excess drug is added to the treatment of naïve samples and the cut point is established using these drug-spiked samples. The method is based on the principle that the excess drug inhibits the availability of preexisting antibodies to bind to the capture/detection system. The competitive inhibition by presence excess drug decreases the assay signal of reactive drug-naive samples. The statistical analysis (target 5% false positive rate) of the immunoinhibition method results in a lower cut point compared to the normal cut-point method. It also improves the detection of clinically significant ADA in clinical studies.





Figure 2: Mechanism of Immuno-inhibition: The spiking of free drug to the pre-existing antibody sample result in the formation of the antibody-drug complex and reduce the availability of antibody binding to capture and detection system.

The critical steps for the immune-inhibition method include:
a) Optimization of minimal drug concentration that results in the highest immunoinhibition
b) Spiking drug to each sample and analyze the drug-spiked sample set
c) Derive cut point using the recommended statistical method
d) Determine the false positive rate of the method
a) Optimization of minimal drug concentration that results in the highest immunoinhibition

The first step of the immunoinhibition method is to define the concentration of the drug required to attain maximal inhibitions without any effect on negative controls. The drug concentration for immunoinhibition is evaluated by spiking the varying amount of drug to positive control and determining the concentration that yields maximal signal inhibition (as shown in figure 3).


Figure 3: Determining optimal drug concentration for the immunoinhibition method (Simhadri et al 2018). In this figure, the anti-SaCas9 (188 ng/mL) and anti-SpCas9 (250 ng/mL) antibodies were pre-incubated with increasing concentrations of SaCas9 (A) and SpCas9 (B) antigen. The highest concentration of antigen used (200 μg/mL) inhibited binding of anti-SaCas9 and anti-SpCas9 antibody

b) Spiking drug to each sample and analyze the drug-spiked sample set
The optimized concentration of a drug is spiked into the drug-naive individual serum and analyzed in the assay. The presence of drugs suppresses the assay signal by binding/saturating preexisting antibodies. As shown in figure 4, individuals without drug spike are randomly dispersed with varying reactivity. In contrast. the same samples with drug spike have homogeneously distributed assay signal. Using this data, the cut point calculated at the 95th percentile from the drug inhibition results in lower cut points and a higher positive rate in the population.
Figure 4: Immunoinhibition Method for Cutpoint determination (Schneider et al. 2018)

c) Derive cut point using the recommended statistical method: Click here for details
d) Determine the false positive rate of the method: Click here for details

Other methods for the cut point calculation includes:
a) Removal of pre-existing antibodies using Protein A/G
b) Titer method

References
Determination of Assay Cut point
Schneider et al. 2016 J Immun Methods
Simhadri et al. 2018 Mol Ther Methods & Clin Dev

CRISPR is a gene editing tools that are being explored as a potential gene therapy candidate for gene deletions, corrections etc. The high efficiency and specificity of the CRISPR enable its global use in basic research and drug discovery research. The CRISPR consist of two components a) Cas9, a protein that has a nuclease activity b) Guide RNA that is recognizes the target sequence and enable cleavage of target sequence catalyzed by Cas9. Cas9 is a protein is originally isolated from bacteria Staphylococcus aureus and Streptococcus pyogenes. Because Cas9 is originated from human pathogenic bacteria, the prior exposure to the protein may results in pre-existing antibodies and T cell response against Cas9. The pre-existing immunogenicity against the Cas9 pose a risk impacting safety and efficacy of CRISPR mediated gene therapy.

Recent studies have reported that the prevalence of the pre-existing antibodies against Cas9 proteins. Simhari et al. 2018 reported the prevalence of anti-Sa Cas9 & anti-Sp Cas9 antibody to be 10.0% and 2.5% respectively using ELISA assay. Another study by Charlesworth et al. 2018 reported higher prevalence of antibodies using immunoblot assay. Although the small sample size, the co-incidence of T-cell response with the pre-existing antibodies are approximately forty six percent (Charlesworth et al. 2018). The different assay methods were used for determining anti-Cas9 antibodies in these studies, and therefore, the outcome should not be compared. Nevertheless, these studies provided the insight on the pre-existing antibody and T cell response against Cas9 protein.

The Cas9 RNA is generally package into the nanoparticle and delivered into the target cells. Alternatively, Cas9 DNA are also packaged into the viral vectors, delivered into the target tissue and expressed endogenously. But the Cas9 protein are rarely administered directly and it may not directly comes in contact with Cas9 antibodies. Therefore, the pre-existing Cas9 antibodies may have lesser impact on drug efficacy and safety. In contrast, the anti-Cas9 IgG antibody and Cas9 activated interferon gamma release (in vitro) suggest a positive of Cas9 specific T-cell and B-cell responses. And the pre-existing Cas9 specific T-cell activity against Cas9 pose a risk of recognizing the MHC- Cas9 transgene complex thereby activating the cytotoxic T lymphocytes and target cell lysis.

Finally these studies have provided the preliminary data on immunogenicity and substantiated the importance of immunogenicity assessment program for drug development that include
a) Characterize pre-existing antidrug antibodies
b) Identify T cell epitopes, HLA restriction
b) Characterization of T cell responses
c) Clinical consequence of pre-existing anti-Cas9 antibodies and T cell responses in safety and efficacy.

References
Charesworth et al. 2018 bioRxiv
Simhadri et al. 2018 Molecular Therapy: Methods & Clinical Development

FDA guidance for Immunogenicity Testing Assays: Development & Validation of Anti-drug Antibody Assays

How to develop reliable, sensitive and specific ADA assays for immunogenicity testing?
The reliable, sensitive and specific ADA assays are crucial for facilitating the understanding of immunogenicity, pharmacokinetics, pharmacodynamics, safety, and efficacy of therapeutic protein products. During development, evaluation of key aspects of the assay such as assay parameters such as sensitivity, specificity, drug tolerance & critical reagent selection and ensure the robust, reproducible and sensitive detection of anti-drug antibodies.

Multiple Choice Question on Haemophilus & Bordetella

1) Which of these small, gram-negative microorganisms are important human pathogens?
a) Haemophilus influenzae
b) Bordetella pertussis
c) Haemophilus parainfluenzae
d) A, B and C

2) …..................was one of the leading bacterial pathogens that caused severe upper respiratory tract infections and even death in children during the nineteenth century, it is now rare in countries where routine vaccination is given to children.
a) Haemophilus influenzae
b) Bordetella pertussis
c) Haemophilus influenzae type b
d) Francisella tularensis

Multiple Choice Question on Mycobacteria tuberculosis and Mycobacteria leprae

1) Mycobacteria are acid-fast positive bacteria because of:
a) the presence of lipopolysaccharide in the bacterial cell wall
b) the presence of mycolic acid in the bacterial cell wall
c) the presence of lipids
d) Both B and C options given above

2) Which of the following first-line antibiotics are usually resistant to Mycobacterium tuberculosis?
a) Isoniazid and ciprofloxacin
b) Isoniazid and Rifampin
c) Rifampin and ciprofloxacin
d) Rifampin and streptomycin

Multiple Choice Question of T-cell Receptors
1) T cells express a transmembrane protein that recognizes the peptide-loaded MHC (pMHC) to activate T cell-mediated immune response. The T-cell receptor is a
a) protein of immunoglobulin superfamily
b) seven transmembrane G protein family
c) tyrosine kinase receptor superfamily
d) None of the above

2) T cell receptor is a heterodimer consist of
a) alpha and beta chain
b) gamma and delta chain
c) Both of the above
d) None of the above

3) Which of the following statement is FALSE regarding the T-cell receptor
a) The alpha and beta chains of TCR are proteins of the immunoglobulin superfamily
b) Both alpha and beta chains consist of one variable domain and one constant domain
c) The TCR resemble Fab fragment of an immunoglobulin
d) The variable region is present in the C terminal region of the TCR